New target for diabetes treatment and prevention

ABSTRACT

The present invention relates to the identification of ALMS1 as the missing player involved in the regulation of the insulin-mediated glucose uptake through GLUT4 sorting vesicles, and to the down-regulation of ALMS1 by αPKC. Accordingly, the present invention relates to a molecule capable of preventing the binding of αPKC on ALMS1 for use for treating or preventing diabetes, in particular type 2 diabetes. In addition, the present invention relates to a method for identifying molecule capable of preventing the binding of αPKC on ALMS1.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/114,080, filed Jul. 26, 2016, now U.S. Pat. No. 10,821,159, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2015/051856, filed Jan. 29, 2015.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Jul. 8, 2016 and is 71 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of the medicine. Moreparticularly, it relates to diabetes.

BACKGROUND OF THE INVENTION

Diabetes mellitus or diabetes is a group of metabolic diseases in whicha person has high blood sugar, either because the pancreas does notproduce enough insulin, or because cells do not respond to the insulinthat is produced.

There are three main types of diabetes:

-   -   Type 1 results from the body's failure to produce insulin, and        currently requires the person to inject insulin or wear an        insulin pump.    -   Type 2 results from insulin resistance, a condition in which        cells fail to use insulin properly.    -   The third one is called gestational diabetes and occurs with        pregnant women.

Rates of type 2 diabetes have increased markedly since 1960 in parallelwith obesity: As of 2010 there are approximately 285 million people withthe disease compared to around 30 million in 1985. Long-termcomplications from high blood sugar can include heart diseases, strokes,diabetic retinopathy, chronic renal failure which may require dialysisand poor circulation in the limbs leading to amputations. Nonketotichyperosmolar coma may occur.

It has been reported that hyperglycemia participates in the onset andprogressive impairment of diabetes mellitus, i.e., glucose toxicitytheory. Namely, chronic hyperglycemia leads to decrease insulinsecretion and further to decrease insulin sensitivity, and as a result,the blood glucose concentration is increased so that diabetes mellitusis self-exacerbated. Therefore, by treating hyperglycemia, theaforementioned self-exacerbating cycle is interrupted so that theprophylaxis or treatment of diabetes mellitus is made possible.

Unfortunately, existing treatments do not succeed in restoringnormoglycaemia in the long term, since beta-cell function declines overtime. Moreover, there is presently no single drug able to reverse allaspects of the disease.

The progressive nature of type 2 diabetes means that many patients willeventually require a combination of oral hypoglycaemic medication,possibly together with insulin and/or exenatide injections.Anti-diabetic agents have been developed in order to counteract the mainmechanisms involved in type 2 diabetes: insulin resistance (biguanidesand thiazolidinediones) and insulin secretion (sulfonylureas, glinides,dipeptidylpeptidase-4 inhibitors, glucagon-like peptide 1 receptoragonists), agents that delay absorption of glucose by gastrointestinaltract or promote weight loss and newer agents that promote renal glucoseexcretion. However, most of these medications have been shown to havedeleterious side effects such as weight gain, peripheral edema orcongestive heart failure and there is a major problem with a loss ofeffectiveness of these agents with long-term use. Thus, despite theincreasing number of therapeutic options for glycaemic control, there isa need for alternative and improved medications for the treatment ofdiabetes and related conditions.

SUMMARY OF THE INVENTION

The inventors identified a new target for treating diabetes, inparticular Type 2 diabetes. They made the novel finding that ALMS1(Alstrom syndrome protein 1) is involved in the regulation by insulin ofglucose absorption by mature adipocytes through its binding interactionswith key molecules involved in regulation of glucose. Briefly, wheninsulin binds its receptor, they showed that a protein complex formsaround Alms1 (the ALMSome) and is activated, leading to H+ pumpactivation, GLUT4 receptor translocation and glucose absorption byadipocytes. They also showed that in the absence of Alms1, and therebyprevention of assembly of the ALMSome, glucose cannot be transportedinto the cells due to a failure of GLUT4 fusion with the cell membrane.Hence, they showed that modulation of ALMS1 complex formation can beused to regulate glucose transport and can thereby be used to circumventinsulin resistance, and treat Type 2 diabetes.

More particularly, the inventors identified two proteins involved inglucose transport regulation by ALMS1, namely TBC1D4 (TBC1 domain familymember 4) and αPKC (PKCα or Protein Kinase C alpha type). Moreparticularly, the binding sites of these two glucose regulating proteinson ALMS1 are so close that the simultaneous binding of both proteins isnot possible due to steric hindrance. TBC1D4, through its interactionwith proteins (i.e., Rab10, Rab14, etc.) and ALMS1, regulates thetranslocation of GLUT4 receptors to the cell membrane. On the otherhand, αPKC, when bound to ALMS1, blocks the TBC1D4 binding site and,thereby down-regulates the translocation of GLUT4 receptors to the cellmembrane, reducing cellular glucose absorption. They furtherdemonstrated that targeting the interaction of ALMS1 and αPKC issufficient to trigger glucose absorption in the adipocytes irrespectiveof the presence of INS. Accordingly, a new therapeutic strategy revealedin this invention is to enhance cellular glucose absorption and reducehyperglycaemia by blocking the binding of αPKC on ALMS1. Mostpreferably, the binding of αPKC on ALMS1 is inhibited in such a way thatthe binding of TBC1D4 on ALMS1 is unaffected or even enhanced.

Accordingly, the present invention relates to a molecule capable ofpreventing the binding of αPKC to ALMS1 for use for treating or delayingthe progression or onset of diabetes mellitus, insulin resistance,diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulinresistance, hyperglycemia, obesity, and hyperinsulinaemia. It alsorelates to the use of such a molecule for the manufacture of amedicament for treating or delaying the progression or onset of diabetesmellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy,diabetic nephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia. It also relates to a method for treating or delayingthe progression or onset of diabetes mellitus, insulin resistance,diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulinresistance, hyperglycemia, obesity, and hyperinsulinaemia, in a subjectin need thereof, wherein a therapeutically effective amount of amolecule capable of preventing the binding of αPKC to ALMS1 isadministered, thereby increasing the glucose absorption induced byinsulin. In a preferred embodiment, the molecule does not interfere withthe binding of TBC1D4 to ALMS1. Preferably, the molecule is selectedfrom the group consisting of peptides or polypeptides or peptidemimetics, antibodies, fragments or derivatives thereof, aptamers,Spiegelmers, and chemical compounds. More preferably, the molecule is apeptide less than 50 amino acids, preferably less than 20 amino acids.

In a first preferred embodiment, the molecule is a peptide comprising anamino acid sequence of a fragment of ALMS1 (SEQ ID NO: 1). Preferably,the molecule is a peptide comprising an amino acid sequence of afragment of ALMS1 including one or several of the residues which arepredicted to mediate the interaction with αPKC, in particular one orseveral of the residues selected in the list consisting of E17, D58,S59, G62, H65, L66, Q736, T737, E738, D828, 5829, T1088, D1089, A1169,Q1170, F2882, L2883, and E2884. In a very particular embodiment, themolecule is a peptide comprising or consisting of one of the followingsequences:

(SEQ ID NO: 5) LDSDSHYGPQHLESIDD; (SEQ ID NO: 6) DSHQTEETL;(SEQ ID NO: 7) QQTLPESHLP; (SEQ ID NO: 8) QALLDSHLPE; (SEQ ID NO: 9)PADQMTDTP; (SEQ ID NO: 10) HIPEEAQKVSAV; (SEQ ID NO: 11) SCIFLEQ,and

-   -   a fragment thereof comprising 6 contiguous amino acids.

In a second preferred embodiment, the molecule is a peptide comprisingan amino acid sequence of a fragment of αPKC (SEQ ID NO: 4). Preferably,the molecule is a peptide comprising an amino acid sequence of afragment of αPKC including one or several of the residues which arepredicted to mediate the interaction with ALMS1, in particular one orseveral of the residues selected in the list consisting of F114, D116,C118, L121, N138, Q142, I145, P148, G433, E545, 5562, 5566, F597, D601,W602, K604, E606, G620, T631, V664, and 1667.

The present invention also relates to a method for identifying moleculessuitable for use for treating or delaying the progression or onset ofdiabetes mellitus, insulin resistance, diabetic retinopathy, diabeticneuropathy, diabetic nephropathy, insulin resistance, hyperglycemia,obesity, and hyperinsulinaemia, wherein the capacity of the molecule toprevent the binding of αPKC to ALMS1 is assayed and the moleculescapable of preventing this binding are selected. The method mayadditionally comprise a step in which the capacity of the selectedmolecule to interfere with the binding of TBC1D4 to ALMS1 is tested andwherein the molecules which do not interfere are selected. Preferably,the binding is determined in a cellular system responsive to insulin.Optionally, the binding is determined in presence and/or absence ofinsulin.

A further therapeutic strategy revealed in this invention is to enhancecellular glucose absorption by enhancing the binding of TBC1D4 on ALMS1.A further therapeutic strategy revealed in this invention is to enhancecellular glucose absorption by upregulating expression of ALMS1.

Accordingly, the present invention further relates to a molecule capableof enhancing the binding of TBC1D4 on ALMS1 or increasing the expressionof ALMS1 for use in treating or delaying the progression or onset ofdiabetes mellitus, insulin resistance, diabetic retinopathy, diabeticneuropathy, diabetic nephropathy, insulin resistance, hyperglycemia,obesity, and hyperinsulinaemia, in particular Type 2 diabetes. It alsorelates to the use of such a molecule for the manufacture of amedicament for treating or delaying the progression or onset of diabetesmellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy,diabetic nephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia, in particular Type 2 diabetes. It also relates to amethod for treating or delaying the progression or onset of diabetesmellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy,diabetic nephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia, in particular Type 2 diabetes, in a subject in needthereof, wherein a therapeutically effective amount of a moleculecapable of enhancing the binding of TBC1D4 on ALMS1 or increasing theexpression of ALMS1 is administered, thereby increasing the glucoseabsorption induced by insulin. In a preferred embodiment, the moleculealso inhibits the binding of αPKC on ALMS1.

The present invention also relates to a method for identifying moleculessuitable for use for treating diabetes, wherein the capacity of themolecule to increase the expression of ALMS1 is assayed and themolecules capable of upregulating ALMS1 are selected. It further relatesto method for identifying molecules suitable for use for treatingdiabetes, wherein the capacity of the molecule to increase the bindingof TBC1D4 to ALMS1 is assayed and the molecules capable of increasingthis binding are selected. Optionally, the method further comprisesdetermining the capacity of the molecule to prevent the binding of αPKCto ALMS1 is assayed and selecting the molecules capable of preventingthis binding

DETAILED DESCRIPTION OF THE INVENTION

The inventors identified ALMS1 as the missing key player involved inregulation the insulin-mediated glucose uptake through GLUT4 sortingvesicles into adipocytes.

It has been now acknowledged that, even if adipose tissue is responsibleof about 20% of the glucose absorption, a dysfunction in this tissue canlead to diabetes occurrence. Therefore, any means capable of regulatingthe insulin-mediated glucose uptake into adipocytes should be able todelay, reverse, or prevent the occurrence of diabetes mellitus, insulinresistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia.

ALMS1 activity is downregulated by the binding of αPKC whereas it isactivated by the binding of TBC1D4. It has also be shown that thebinding sites of these two proteins on ALMS1 are so close that thesimultaneous binding of both proteins is not allowed due to sterichindrance. Therefore, this regulation mechanism is a new target fortreating or delaying the progression or onset of diabetes mellitus,insulin resistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia and the inventors propose to use a molecule capable ofpreventing the binding of αPKC to ALMS1 for these therapeuticindications.

Definitions

ALMS1, Alstrom syndrome protein 1, is a protein encoded by the ALMS1gene. Mutations in the ALMS1 gene have been found to be causative forAlström syndrome. It is described in several databases, namely UniProtID No Q8TCU4; Gene ID No 7840, HGNG ID No 428. Reference sequences aredisclosed in Genbank under NM_015120.4 for mRNA and NP_055935.4 forprotein. The protein sequence of human ALMS1 is disclosed in SEQ ID NO:1.

TBC1D4 (TBC1 domain family member 4), also currently called As160, issupposed to act as a GTPase-activating protein for RAB2A, RAB8A, RAB10and RAB14. It is described in several databases, namely UniProt ID No060343, Gene ID No 9882, HGNG ID No 19165. Reference sequences aredisclosed in Genbank under NM_014832.3 for mRNA and NP_055647.2 forprotein (for isoform 1, chosen as canonical sequences). The isoform 2,which differs from isoform by the missing of the amino acids inpositions 678-740 and referenced in UniProt under No 060343-2, promotesinsulin-induced glucose transporter SLC2A4/GLUT4 translocation at theplasma membrane, thus increasing glucose uptake. The protein sequence ofhuman TBC1D4 (isoform 1) is disclosed in SEQ ID NO: 2. The proteinsequence of human TBC1D4 (isoform 2) is disclosed in SEQ ID NO: 3.

Protein kinase C alpha type, also called αPKC, PKC-A or PKC-alpha,belongs to a family of serine- and threonine-specific protein kinasesthat can be activated by calcium and the second messengerdiacylglycerol. It is described in several databases, namely UniProt IDNo P17252, Gene ID No 9393, HGNG ID No 5578. Reference sequences aredisclosed in Genbank under NM_02737.2 for mRNA and NP_002728.1 forprotein. The protein sequence of human αPKC is disclosed in SEQ ID NO:4.

Screening Methods

The present invention relates to an in vitro or ex vivo method foridentifying, screening or selecting a molecule capable of preventing thebinding of αPKC to ALMS1. The method comprises determining the effect ofmolecule(s) on the binding of αPKC to ALMS1, and selecting themolecule(s) if the binding of αPKC to ALMS1 is decreased or prevented.Preferably, the method further comprises determining the effect ofmolecule(s) on the binding of TBC1D4 to ALMS1, and eliminating themolecule(s) if the binding of TBC1D4 to ALMS1 is decreased or prevented.Optionally, the method may comprise a step of selecting the molecule(s)if the binding of TBC1D4 to ALMS1 is increased or enhanced.

The present invention also relates to an in vitro or ex vivo method foridentifying, screening or selecting a molecule capable of enhancing orincreasing the binding of TBC1D4 to ALMS1. The method comprisesdetermining the effect of molecule(s) on the binding of TBC1D4 to ALMS1,and selecting the molecule(s) if the binding of TBC1D4 to ALMS1 isincreased or enhanced. Optionally, the method further comprisesdetermining the effect of molecule(s) on the binding of αPKC to ALMS1,and selecting the molecule(s) if the binding of αPKC to ALMS1 isdecreased or prevented.

In order to determine the effect of a molecule on the binding of αPKCand/or TBC1D4 to ALMS1, any technology known by the person skilled inthe art can be carried out, in particular any method suitable fordetermining protein interactions. For example, recombinant or purifiednative ALMS1 or αPKC can be bound to a surface plasmon resonance chipand the other molecule flowed over the chip to assess the bindingaffinity, for example in a Biacore (General Electric, USA) machine. Thesame approach can be used to measure the binding affinity of ALMS1 andTBC1D4 or of ALMS1 and αPKC.

The effect of molecule(s) on the binding of αPKC and/or TBC1D4 to ALMS1is determining by measuring the binding of αPKC and/or TBC1D4 to ALMS1in absence and in presence of the tested molecule and by comparing thebindings of αPKC and/or TBC1D4 to ALMS1.

In addition, the screening method may comprise a preliminary step forselecting the molecule(s) capable of binding to ALMS1. Indeed, it couldbe advantageous that the molecule preventing the interaction betweenALMS1 and αPKC acts directly on the ALMS1 binding site for αPKC.

Alternatively, the screening method may comprise a preliminary step forselecting the molecule(s) capable to bind to αPKC. Indeed, it could alsobe advantageous that the molecule preventing the interaction betweenALMS1 and αPKC acts directly on the αPKC binding site for ALMS1.

In addition, the screening method may comprise a preliminary step forselecting the molecule(s) capable to bind to TBC1D4.

In a preferred embodiment for identifying, screening or selecting amolecule capable of preventing the binding of αPKC to ALMS1, thescreening method of the present invention further comprises determiningthe effect of the molecule(s), in particular the selected molecule(s),on the binding of TBC1D4 to ALMS1 and selecting the molecule(s) if thebinding of TBC1D4 to ALMS1 is not decreased or prevented by themolecule(s). Even more, the method may comprise a step of selecting themolecule(s) if TBC1D4 to ALMS1 is increased or enhanced by themolecule(s).

In a preferred embodiment for identifying, screening or selecting amolecule capable of enhancing the binding of TBC1D4 to ALMS1, thescreening method of the present invention further comprises determiningthe effect of the molecule(s), in particular the selected molecule(s),on the binding of αPKC to ALMS1 and selecting the molecule(s) if thebinding of αPKC to ALMS1 is decreased or prevented by the molecule(s).

Due to the large size of the binding partners, in particular ALMS1 andTBC1D4, the inventors prefer using cellular systems for the screeningmethods. Preferably, the cellular system is a cellular system responsiveto insulin. For instance, the cellular system could be selected among amesenchymal cell line, a mesenchymal stem cell, an adipose mesenchymalstem cell, a pre-adipocyte and an adipocyte. Preferably, the cell is ahuman cell.

Then, the binding determinations can be carried in absence or presenceof insulin, preferably in presence of insulin for the binding of αPKC toALMS1 and in the presence insulin for the binding of TBC1D4 to ALMS1.

In a first aspect, immunoprecipitation assay using ALMS1 as bait can becarried, in particular as detailed in the experimental section. Forinstance, the assay can be carried out with cells, in particularadipocytes, cultured in absence and/or presence of insulin, preferablyin absence of insulin. The molecules to be tested are added in theculture medium. Then, αPKC is immunodetected. Optionally, TBC1D4 canalso be immunodetected. This method is disclosed in details in theExamples section.

In a preferred embodiment, the amount of αPKC bound to ALMS1 isdetermined and compared to the amount in absence of tested molecules, inparticular in absence of insulin. If the amount of αPKC bound to ALMS1decreases in presence of the tested molecule, then the molecule isselected.

The amount of TBC1D4 bound to ALMS1 is determined and compared to theamount in absence of tested molecules, in particular in presence ofinsulin or both in presence and absence of insulin. If the amount ofTBC1D4 bound to ALMS1 decreases in presence of the tested molecule, thenthe molecule is rejected. If the amount of TBC1D4 bound to ALMS1increases in presence of the tested molecule, then the molecule isselected.

In a second aspect, affinity purification coupled to mass spectrometrycan be carried out, in particular after chemical crosslinking. Forinstance, cells may be cultured in a medium devoid of methionine andleucine but comprising photo-activable methionine and leucine. Then,cells are UV irradiated in order to stabilize protein complexes andprotein complexes are analyzed by mass spectrometry.

Other methods are available to the person skilled in the art, e.g.,Biomolecular fluorescence complementation, Tandem affinity purification,and the like. In particular, WO2012/117245 discloses a method foridentifying molecules capable of preventing the interaction between twoproteins: WO2012/117245 (i.e., for identifying small molecules).WO12174489 also discloses methods for developing molecules suitable forpreventing interaction between two proteins.

In addition, suitable molecules can also be designed by molecularmodelling. Such methods are for instance detailed in the Examplesection.

In a particular aspect, the present invention relates to a structuralhomology model of ALMS1 and its use in an in silico method to identifymolecules able to inhibit or stimulate ALMSome function, in particularto inhibit the interaction between ALMS1 and αPKC and/or to increase theinteraction between ALMS1 and TBC1D4.

It also relates to a structural homology model of TBC1D4 and its use inan in silico method to identify molecules able to inhibit or stimulateALMSome function, in particular to increase the interaction betweenALMS1 and TBC1D4.

The present invention also relates to a method for identifying,screening or selecting a molecule capable of upregulating ALMS1 at thegene and protein level. The method comprises determining the effect ofmolecule(s) on the expression of ALMS1, and selecting the molecule(s) ifthe expression of ALMS1 is increased. In order to determine the effectof a molecule on the expression of ALMS1, any technology known by theperson skilled in the art can be carried out. Various techniques knownin the art may be used to detect or quantify ALMS1 expression, includingsequencing, hybridisation, amplification and/or binding to specificligands (such as antibodies). Suitable methods include Southern blot(for DNAs), Northern blot (for RNAs), fluorescent in situ hybridization(FISH), gel migration, ELISA, radio-immunoassays (MA) andimmuno-enzymatic assays (IEMA).

By “increased”, “increase” or “enhance” is intended to refer to abinding increased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% whencompared to the binding measured in absence of the tested molecule inthe same conditions. By “decreased” or “decrease” is intended to referto a binding decreased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90%when compared to the binding measured in absence of the tested moleculein the same conditions.

In addition, the screening methods of the present invention may compriseassay with animal models. Molecules to be tested may be administered tothe animal models and the effect of the molecules on the glucoseabsorption or diabetes could be assessed. For instance, the animalmodels could be mice or rat with insulin resistance, diabetes, orhyperglycemia. The effect of the molecule can be assessed by measuringthe level of blood glucose.

Molecules

The molecules capable of preventing or blocking the binding of αPKC toALMS1 can be any ligand capable of binding either αPKC or ALMS1 and,thereby preventing or blocking the binding of αPKC to ALMS1.

In a first aspect, the present invention relates to a molecule thatprevents or blocks the binding of αPKC to ALMS1 by interacting with oneor more of the ALMS1 residues selected in the list consisting of E17,D58, S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089,A1169, Q1170, F2882, L2883, and E2884. In an alternative aspect, thepresent invention relates to a molecule that prevents or blocks thebinding of αPKC to ALMS1 by interacting with one or more of the αPKCresidues selected in the list consisting of F114, D116, C118, L121,N138, Q142, I145, P148, G433, E545, S562, S566, F597, D601, W602, K604,E606, G620, T631, V664, and 1667.

The molecules capable of enhancing or increasing the binding of TBC1D4to ALMS1 can be any ligand capable of binding either TBC1D4 or ALMS1and, thereby enhancing or increasing the binding of TBC1D4 to ALMS1.

In a first aspect, the present invention relates to a molecule thatenhances or increases the binding of TBC1D4 to ALMS1 by interacting withone or more of the ALMS1 residues selected in the list consisting ofH65, L66 and S2879. In an alternative aspect, the present inventionrelates to a molecule that enhances or increases the binding of TBC1D4to ALMS1 by interacting with one or more of the TBC1D4 residues selectedin the list consisting of G75, A76, P77, A78, R80, E81, V82, and 183.

The present invention relates to such molecules, a pharmaceuticalcomposition comprising such molecules, and the use of such molecules asa drug or for the manufacture of a drug.

The molecules can be peptides or polypeptides or peptide mimetics,antibodies, fragments or derivatives thereof, aptamers, Spiegelmers, orchemical compounds. The molecules can be selected by the screeningmethods known in the art or as detailed above and can be designed by anyconvenient in silico modeling method.

In a preferred embodiment, the molecule is a peptide or polypeptide.Preferably, the peptide may have between 5 and 50 amino acids. Morepreferably, it has between 5 and 20 amino acids. More preferably, thepeptide or polypeptide comprises less than 50 amino acids, morepreferably less than 40, 30, 20, 15 or 10 amino acids.

In a first aspect, the molecule is a peptide or polypeptide comprisingan amino acid sequence of a fragment of ALMS1 (SEQ ID NO: 1). In apreferred embodiment, the molecule is a peptide or polypeptidecomprising an amino acid sequence of a fragment of ALMS1 including oneor several of the residues which are predicted to mediate theinteraction with αPKC. In particular, these residues are selected in thelist consisting of E17, D58, S59, G62, H65, L66, Q736, T737, E738, D828,5829, T1088, D1089, A1169, Q1170, F2882, L2883, and E2884. Morepreferably, these residues are selected in the list consisting of D58,S59, G62, H65, L66, Q736, T737, E738, D828, S829, T1088, D1089, A1169,Q1170, F2882, L2883, and E2884. D58, S59, G62, H65 and L66 define afirst interaction segment. T737 and E738 define a second interactionsegment. D828 and S829 define a third interaction segment. T1088 andD1089 define a fourth interaction segment. A1169 and Q1170 define afifth interaction segment. F2882, L2883 and E2884 define a sixthinteraction segment.

In a very particular aspect, the peptide or polypeptide comprises orconsists of one of the following sequences:

-   -   LDSDSHYGPQHLESIDD (SEQ ID NO: 5), targeting the first        interaction segment;    -   DSHQTEETL (SEQ ID NO: 6), targeting the second interaction        segment;    -   QQTLPESHLP (SEQ ID NO: 7);    -   QALLDSHLPE (SEQ ID NO: 8), targeting the third interaction        segment;    -   PADQMTDTP (SEQ ID NO: 9), targeting the fourth interaction        segment;    -   HIPEEAQKVSAV (SEQ ID NO: 10), targeting the fifth interaction        segment;    -   SCIFLEQ (SEQ ID NO: 11), targeting the sixth interaction        segment, and    -   a fragment thereof comprising 6 contiguous amino acids.

In a second aspect, the molecule is a peptide or polypeptide comprisingan amino acid sequence of a fragment of αPKC (SEQ ID NO: 4). In apreferred embodiment, the molecule is a peptide or polypeptidecomprising an amino acid sequence of a fragment of αPKC including one orseveral of the residues which are predicted to mediate the interactionwith ALMS1. In particular, these residues are selected in the listconsisting of F114, D116, C118, L121, N138, Q142, I145, P148, G433,E545, 5562, 5566, F597, D601, W602, K604, E606, G620, T631, V664, and1667. F114, D116, C118 and L121 may define a first interaction segment.N138, Q142, I145 and P148 may define a second interaction segment. E545,5562 and 5566 may define a third interaction segment. F597, D601, W602,K604, and E606 define a fourth interaction segment. V664 and 1667 maydefine a fifth interaction segment.

Optionally, the peptide or polypeptide may comprise one, two, three,four or five amino acid substitution in comparison to the referencesequence, i.e., SEQ ID NO: 1 for peptides derived from ALMS1, SEQ ID NO:4 for peptides derived from αPKC, and SEQ ID NO: 2 or 3 for peptidesderived from TBC1D4.

The peptide or polypeptide may further comprise a moiety facilitatingits cellular uptake or entry, in particular a PTD (protein transductiondomain). PTD generally comprises a certain amino acid sequence of 10 to20 amino acids (Matsushita and Matsui, (2005), J Mol Med 83, 324-328;Vivès et al, Biochimic et Biophysica Acta, 2008, 1786, 126-138). PTD ismainly composed of basic amino acids such as arginine or lysine, andrepresentative examples of the PTD include arginine rich peptides suchas poly R₈ (RRRRRRRR) (SEQ ID NO: 18) or (RRPRRPRRPRRPRRP) (SEQ ID NO:19), antennapedia or penetratin peptide such as (RQIKIWFQNRRMKWKK) (SEQID NO: 20) or HIV-Tat (YGRKKRRQRRR) (SEQ ID NO: 21).

In a particular aspect, the molecule is an antibody, fragment orderivative thereof.

The peptide or polypeptide can be made of natural amino acids and/orunnatural amino acids. By “unnatural amino acids” is intended an analogor derivative of a natural amino acid (i.e., Ala, Val, Gly, Leu, Ile,Lys, Arg, Glu, GLn, Asp, Asn, His, Tyr, Phe, Trp, Ser, Pro, Thr, Cys,Met). They present a modified side chain, e.g. shorter, longer or withdifferent functional groups. Isomers D and L are contemplated, inparticular because isomers D are not sensible to proteases. In addition,modifications in some or all peptide bounds are also contemplated inorder to increase the proteolysis resistance, in particular by (—CO—NH—)by (—CH₂—NH—), (—NH—CO—), (—CH₂—O—), (—CH₂—S—), (—CH₂—CH₂—), (—CO—CH₂—),(—CHOH—CH₂—), (—N═N—), and/or (—CH═CH—). The peptide can present acarboxylic C terminal end (—COO⁻) and an amide one (—CONH₂). The peptidecan also be D-retro-inverso sequence of a peptide as disclosed herein.The N terminal can be modified, especially with an acetyl radical.Optionally, the peptide or polypeptide can be PEGylated in order toincrease the stability. Alternatively, the peptide can be modified tobecome a stapled peptide. The term “stapled peptide” as used hereinrefers to artificially modified peptide in which the structure isstabilized with one or more artificial molecular bridging (cross links)that connects adjacent turns of α-helices in the peptide. The modalitiesfor preparing stapled peptides have been reviewed extensively forinstance in Verdine & Hilinski (2012, Methods Enzymol, 503, 3-33),WO10033617 and WO10011313, the disclosure of which being incorporatedherein by reference.

The present invention further relates to a pharmaceutical compositioncomprising a peptide as defined above and a pharmaceutically acceptablecarrier/excipient. It also relates to a peptide as defined above for useas a drug or to the use of a peptide as defined above for themanufacture of a medicament.

In an alternative embodiment, the molecule is an antibody, a fragmentthereof or a derivative thereof. As used herein, the terms “antibody”and “immunoglobulin” have the same meaning and are used indifferently inthe present invention. The term “antibody” refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen-binding site thatimmunospecifically binds an antigen. Antibodies include any kind ofantibodies, preferably monoclonal. They can be for instance IgG(immunoglobulin G) or VHH (heavy chain variable domain antibody fromcamelids). Antibodies fragments or derivatives thereof include Fab,Fab′, F(ab′)2, scFv, (scFv)2, dAb, complementarity determining region(CDR) fragments, linear antibodies, single-chain antibody molecules,minibodies, diabodies, and multispecific antibodies formed from antibodyfragments.

Antibodies, fragments or derivatives can block the interaction betweenALMS1 and αPKC. Preferably, they have no effect on the interactionbetween ALMS1 and TBC1D4 or have an enhancing effect on the interaction.

In a first embodiment, the antibody is specific for ALMS1. Inparticular, the epitope of the antibody comprises one or several of theALMS1 residues involved in the interaction with αPKC, in particular oneor several residues selected in the list consisting of E17, D58, S59,G62, H65, L66, Q736, T737, E738, D828, 5829, T1088, D1089, A1169, Q1170,F2882, L2883, and E2884.

Alternatively, the antibody is specific for αPKC. In particular, theepitope of the antibody comprises one or several of the αPKC residuesinvolved in the interaction with ALMS1, in particular one or severalresidues selected in the list consisting of F114, D116, C118, L121,N138, Q142, I145, P148, G433, E545, 5562, 5566, F597, D601, W602, K604,E606, G620, T631, V664, and 1667.

Such antibodies can be produced by immunizing non-human mammals withimmunogenic peptides or proteins comprising one or several residuesidentified as involved in the interaction between ALMS1 and αPKC.Alternatively, library of antibodies can be provided and screened.Produced antibodies, fragments or derivatives are then screened fortheir capacity to bind one of the interacting partners and/or theircapacity to prevent, inhibit or block the interaction between ALMS1 andαPKC. In addition, as previously specified, antibodies, fragments orderivatives can be further screened for their capacity to modulate theinteraction between TBC1D4 and ALMS1, and selected if they increase orenhance the interaction.

Antibodies, fragments or derivatives can enhance the interaction betweenALMS1 and TBC1D4. Preferably, they have a blocking effect on theinteraction between ALMS1 and αPKC.

In a first embodiment, the antibody is specific for ALMS1. Inparticular, the epitope of the antibody comprises one or several of theALMS1 residues involved in the interaction with TBC1D4, in particularone or several residues selected in the list consisting of H65, L66 andS2879.

Alternatively, the antibody is specific for TBC1D4. In particular, theepitope of the antibody comprises one or several of the TBC1D4 residuesinvolved in the interaction with ALMS1, in particular one or severalresidues selected in the list consisting of G75, A76, P77, A78, R80,E81, V82, and 183.

Such antibodies can be produced by immunizing non-human mammals withimmunogenic peptides or proteins comprising one or several residuesidentified as involved in the interaction between ALMS1 and TBC1D4.Alternatively, library of antibodies can be provided and screened.Produced antibodies, fragments or derivatives are then screened fortheir capacity to bind one of the interacting partners and/or theircapacity to enhance or increase the interaction between ALMS1 andTBC1D4. In addition, as previously specified, antibodies, fragments orderivatives can be further screened for their capacity to modulate theinteraction between αPKC and ALMS1, and selected if they decrease orblock the interaction.

The preparation of monoclonal or polyclonal antibodies is well known inthe art, and any of a large number of available techniques can be used(see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy (1985)). Techniques for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce antibodies to desired polypeptides. Also, transgenic mice, orother organisms such as other mammals, may be used to express humanized,chimeric, or similarly-modified antibodies. Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)).

For aptamers and Spiegelmers, similar methods can be used in order toselect aptamers and Spiegelmers. These methods are well-known by theperson skilled in the art.

As used here, the term “aptamer” means a molecule of nucleic acid or apeptide able to bind ALMS1, αPKC or TBC1D4. It refers to a class ofmolecule that represents an alternative to antibodies in term ofmolecular recognition. Aptamers are oligonucleotide or oligopeptidesequences with the capacity to recognize virtually any class of targetmolecules with high affinity and specificity.

Such ligands may be isolated through Systematic Evolution of Ligands byEXponential enrichment (SELEX) of a random sequence library, asdescribed in Tuerk C. and Gold L., Science, 1990, 249(4968):505-10. Therandom sequence library is obtainable by combinatorial chemicalsynthesis of DNA. In this library, each member is a linear oligomer,eventually chemically modified, of a unique sequence. Possiblemodifications, uses and advantages of this class of molecules have beenreviewed in Jayasena S. D., Clin. Chem., 1999, 45(9):1628-50. Peptideaptamers consist of a conformationally constrained antibody variableregion displayed by a platform protein, such as E. coli Thioredoxin Athat are selected from combinatorial libraries by two hybrid methods(Colas et al., Nature, 1996, 380, 548-50).

Spiegelmers have been disclosed for instance in WO 98/08856. They aremolecules similar to aptamers. However, spiegelmers consist eithercompletely or mostly of L-nucleotides rather than D-nucleotides incontrast to aptamers. Otherwise, particularly with regard to possiblelengths of spiegelmers, the same applies to spiegelmers as outlined inconnection with aptamers.

Chemical compounds refers to a molecule of less than about 1500 Daltons,1000 Daltons, 800 Daltons, or even less than about 500 Daltons, inparticular organic or inorganic compounds. Structural design inchemistry should help to find such a molecule. The molecule may havebeen identified by a screening method disclosed in the presentinvention.

Synthetic compound libraries are commercially available from a number ofcompanies including Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). Combinatorial libraries are availableor can be prepared according to known synthetic techniques.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available from e.g., PanLaboratories (Bothell, Wash.) and MycoSearch (NC), or are readilyproducible by methods well known in the art.

Additionally, natural and synthetically produced libraries and compoundscan be further modified through conventional chemical and biochemicaltechniques.

The molecule can be linked, covalently or not, to a moiety targeting therelevant tissues, preferably the adipose or to a moiety facilitating theentrance of the molecule into cells.

Therapeutic Indications

The inventors propose to use the molecules as disclosed herein forincreasing the glucose uptake, in particular by adipocytes, therebyregulating or controlling the blood glucose level. Then, the moleculesare suitable for treating or delaying the progression or onset ofdiabetes mellitus, insulin resistance, diabetic retinopathy, diabeticneuropathy, diabetic nephropathy, insulin resistance, hyperglycemia,obesity, and hyperinsulinaemia.

Diabetes mellitus is characterized by hyperglycemia. More particularly,type 2 diabetes is characterized by hyperglycemia and insulinresistance. Obesity is thought to be the primary cause of type 2diabetes in people who are genetically predisposed to the disease.Diabetic retinopathy, diabetic neuropathy, diabetic nephropathy arewell-known disorders associated with diabetes and insulin resistance.

Then, decreasing the glycemia by increasing the glucose uptake couldtreat or delay the progression or onset of these diseases.

The present invention also relates to the molecules according to theinvention for use for reducing the dose of insulin or stopping theinsulin treatment when used for treating diabetes in a subject, to theuse of the molecules according to the invention for the manufacture of amedicament for reducing the dose of insulin or stopping the insulintreatment when used for treating diabetes in a subject, or to a methodfor treating diabetes in a subject, wherein a therapeutically effectiveamount of a molecule according to the invention is administered to asubject with a decreased dose of insulin or in absence of insulintreatment. More generally, it can be used to lower the doses ofanti-diabetic drugs.

By “treat” or “treatment” is intended that the disease is cured,alleviated or delayed. It includes the preventive or curative treatment.The term treatment designates in particular the correction, retardation,or reduction of an impaired glucose homeostasis. The term “treatment”also designates an improvement in glucose uptake (e.g., capture ofglucose by adipocytes). Within the context of the invention, the terms“controlling the blood glucose level” or “the control of blood glucoselevel” refer to the normalization or the regulation of the blood orplasma glucose level in a mammalian subject having abnormal levels(i.e., levels that are below or above a known reference, median, oraverage value for a corresponding mammalian subject with a normalglucose homeostasis).

The present invention relates to the pharmaceutical or veterinary use ofthe molecule. Accordingly, the subject may be any mammal, preferably ahuman subject, such as an adult or a children. In a particularembodiment, the subject is a subject suffering of obesity. Optionally,the subject has no detectable anti-islet antibodies, and ultrasonographyrevealed no pancreatic abnormalities. In the context of a veterinaryapplication, the subject can be an animal, preferably a mammal, inparticular a pet animal such as a dog, a cat or a horse.

The molecules according to the invention can be used in combination withone or more additional active drugs, preferably anti-diabetic drugs, inparticular for treating or delaying the progression or onset of diabetesmellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy,diabetic nephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia.

Therefore, the present invention also relates to a pharmaceuticalcomposition comprising a molecule according to the present invention andone or more additional active drugs, preferably an anti-diabetic drug.

It further relates to a product or kit containing a molecule accordingto the invention and one or more additional active drugs, preferablyanti-diabetic drugs, as a combined preparation for simultaneous,separate or sequential use, or a combined preparation which comprises amolecule according to the invention and one or more additional activedrugs, preferably anti-diabetic drugs, for simultaneous, separate orsequential use, in particular for treating or delaying the progressionor onset of diabetes mellitus, insulin resistance, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, insulin resistance,hyperglycemia, obesity, and hyperinsulinaemia.

It relates to a molecule according to the invention for use for treatingor delaying the progression or onset of diabetes mellitus, insulinresistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia in combination with one or more additional activedrugs, preferably anti-diabetic drugs.

It further relates to the use of a molecule according to the inventionand one or more additional active drugs, preferably anti-diabetic drugs,for the manufacture of a medicament, in particular treating or delayingthe progression or onset of diabetes mellitus, insulin resistance,diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulinresistance, hyperglycemia, obesity, and hyperinsulinaemia.

Finally, it relates to a method for treating or delaying the progressionor onset of diabetes mellitus, insulin resistance, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, insulin resistance,hyperglycemia, obesity, and hyperinsulinaemia, wherein a therapeuticeffective amount of a molecule according to the invention isadministered in combination with a therapeutic or sub-therapeuticeffective amount of one or more additional active drugs, preferablyanti-diabetic drugs. By “sub-therapeutic” is intended to refer to anamount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10% of theconventional therapeutic dosage (in particular for the same indicationand the same administration route).

In particular, the additional active drug is a drug used for treating ordelaying the progression or onset of diabetes mellitus, insulinresistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia. For instance, the additional drug can be ananti-diabetic drug such as a hypoglycemic agent or an antihyperglycemicagent. It may be selected in the non-exhaustive list comprising insulin,metformin, sulfonylureas such as tolbutamide, acetohexamide, tolazamide,chlorpropamide, glyburide (also called glibenclamide), glimepiride,glipizide, glicazide, glycopyramide and gliquidone, alpha-glucosidaseinhibitors such as acarbose, miglitol and voglibose, thiazolidinedionessuch as pioglitazone and rosiglitazone, a meglitinide such asrepaglinide and nateglinide, incretin mimetics, glucagon-like peptideanalogs and agonists such as exenotide, taspoglutide and liraglutide,dipeptidyl peptidase-4 inhibitors such as vildagliptin, sitagliptin,saxagliptin, linagliptin, allogliptin, and septagliptin, amylin analogssuch as pamlintide, glycourics such as canagliflozin and dapagliflozin,or any combination thereof. The form of the pharmaceutical compositions,the route of administration, the dosage and the regimen naturally dependupon the condition to be treated, the severity of the illness, the age,weight, and sex of the patient, etc.

The pharmaceutical or therapeutic compositions of the invention can beformulated for a topical, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous or intraocular administration and the like.

The molecule used in the pharmaceutical composition of the invention ispresent in a therapeutically effective amount. The term “therapeuticallyeffective amount” as used in the present application is intended anamount of therapeutic agent, administered to a patient that issufficient to constitute a treatment of diabetes mellitus, insulinresistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia as defined above.

The pharmaceutical composition comprising the molecule is formulated inaccordance with standard pharmaceutical practice (Lippincott Williams &Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known bya person skilled in the art.

For oral administration, the composition can be formulated intoconventional oral dosage forms such as tablets, capsules, powders,granules and liquid preparations such as syrups, elixirs, andconcentrated drops. Non toxic solid carriers or diluents may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, talcum, cellulose,glucose, sucrose, magnesium, carbonate, and the like. For compressedtablets, binders, which are agents which impart cohesive qualities topowdered materials, are also necessary. For example, starch, gelatine,sugars such as lactose or dextrose, and natural or synthetic gums can beused as binders. Disintegrants are also necessary in the tablets tofacilitate break-up of the tablet. Disintegrants include starches,clays, celluloses, algins, gums and crosslinked polymers. Moreover,lubricants and glidants are also included in the tablets to preventadhesion to the tablet material to surfaces in the manufacturing processand to improve the flow characteristics of the powder material duringmanufacture. Colloidal silicon dioxide is most commonly used as aglidant and compounds such as talc or stearic acids are most commonlyused as lubricants.

For transdermal administration, the composition can be formulated intoointment, cream or gel form and appropriate penetrants or detergentscould be used to facilitate permeation, such as dimethyl sulfoxide,dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginalsuppositories can be used. The active compound can be incorporated intoany of the known suppository bases by methods known in the art. Examplesof such bases include cocoa butter, polyethylene glycols (carbowaxes),polyethylene sorbitan monostearate, and mixtures of these with othercompatible materials to modify the melting point or dissolution rate.

Pharmaceutical compositions according to the invention may be formulatedto release the active drug substantially immediately upon administrationor at any predetermined time or time period after administration.

Pharmaceutical compositions according to the invention can comprise oneor more molecule of the present invention associated withpharmaceutically acceptable excipients and/or carriers. These excipientsand/or carriers are chosen according to the form of administration asdescribed above.

In a particular embodiment, the pharmaceutical composition according tothe invention comprises 0.001 mg to 10 g of the molecule of theinvention. Preferably, pharmaceutical composition according to theinvention comprises 0.01 mg to 1 g of the molecule of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Metabolic characterization of the Alms^(foz/foz) mice

(A) Mean body weight of WT and Alms^(foz/foz) male mice (n=6-8 mice pergenotype). (B) Photograph of visceral adipose tissue from WT andAlms^(foz/foz) Scale bar: 25 μM. (C) Insulin tolerance test (I.T.T.)performed on WT and Alms^(foz/foz) mice and the corresponding histogramshowing the Area under the curve (A.U.C.) for each genotype (n=6-8 miceper group). p<0.001). (D) Mean body weight of WT and Alms^(foz/foz) malemice (n=6-8 mice per genotype). (E) Photograph of visceral adiposetissue from corresponding WT and Alms^(foz/foz) Scale bar: 25 μM. (F)Insulin tolerance test performed on WT and Alms^(foz/foz) mice and thecorresponding histogram showing the A.U.C. for each genotype (n=6-8 miceper group). *** stands for p-value<0.001. (G) Immunoblots for theindicated proteins in insulin sensitive tissues from nonobese WT andAlms^(foz/foz) mice. (H) Results of radioactive counts in differenttarget tissues after injection of radioactive deoxyglucose to WT andAlms^(foz/foz) mice (n=5 mice per genotype). * stands for p-value=0.05.

FIG. 2. ALMS1 silencing effect in human mature adipocytes

(A) Photographs showing the lack of absorption of 2-NBDG (green) incontrol (shCTRL shRNA) or ALMS1-deprived adipocytes (ALMS1 shRNA)silenced mature adipocytes in absence of INS. (B) Photographs depictinglack of absorption of 2-NBDG in ALMS1 shRNA compared to CTRLshRNA.Nuclei were counterstained with DAPI, DIC: Differential InterferenceContrast pictures. (C) 3D images of CTRLshRNA or ALMS1shRNA matureadipocytes stained for intracellular Triglycerides (TG), plasma membranein red (PM) and nuclei in blue (DAPI). (D) Measurements of fluorescentlevels correlating with amounts of intracellular TG in mature adipocytes(n=16 wells per condition measured) *p-value=0.05. (E-F) Immunodetectionof AKT and pS473-AKT in CTRLshRNA and ALMS1shRNA treated matureadipocytes in presence and absence of INS. (G) 3D images of CTRLshRNAand ALMS1shRNA mature adipocytes showing cellular localization ofInsulin receptor (IR in red) and GLUT4 (in green) in absence of Ins.Cut-view images displaying the dynamics of GLUT4 localization in absenceof Ins. (H), after 30 min. INS. stimulation (I) and with 30 min INS.stimulation followed by 2 hours of absence of INS. (J) in CTRLshRNA andALMS1shRNA mature adipocytes. Scale bars: 25 μm in A, B, C and 5 μm inG-J.

FIG. 3. Predicted interaction sites on ALMS1 protein and modelling ofits partner TBC1D4.

(A) Predicted 3D structure of the ALMS1 protein with helices and loops.(B) Predicted 3D structure of the ALMS1 protein with the potentialinteracting sites represented by red dots. (C) Primary sequence ofTBC1D4 protein with indicated localization of binding sites orinteracting domains. (D) Predicted 3D structure of the TBC1D4 protein.

FIG. 4. ALMS1 is required for TBC1D4 cellular trafficking

(A) In silico predicted 3D structure showing spatial interaction betweenALMS1 and TBC1D4 with an enlarged view of the interaction sitehighlighting the predicted interacting amino acid residues (L66, Y61 andS2879) of the ALMS1 protein. (B) 3D image from immunostained matureadipocytes depicting co-localization of TBC1D4 (green) and ALMS1 (red).Nuclei were counterstained with DAPI (blue). (C-D) Immunoblots for theindicated proteins on cell lysates (50 μg total protein loaded per lane)for CTRLshRNA and ALMS1shRNA mature adipocytes treated with or withoutinsulin. 3D images of immunofluorescence experiments performed on eitherCTRLshRNA or ALMS1shRNA or TBC1D4shRNA mature adipocytes depictingcellular localization of GLUT4 in absence of Insulin (−INS) (E) or inthe presence of INS. (F). PM: Plasma membrane and nuclei counterstainedwith DAPI. 3D images of immunofluorescence experiments performed oneither CTRLshRNA or ALMS adipocytes showing cellular localization ofGLUT4 (green) and TBC1D4 in absence of INS (G) or when treated 30 min.with INS. (H). Scale bars: 10 μm.

FIG. 5. TBC1D4 is not the sole interacting partner of ALMS1 playing arole in the adipocyte biology

(A-C) Photographs showing absorption of 2-NBDG in either CTRLshRNA orALMS1shRNA or TBC1D4shRNA deprived adipocytes after 30 min Ins.stimulation. (D-F) 3D images obtained using non-permeablized fixatedmature adipocytes stimulated with INS. following immunodetection ofGLUT4 membrane bound (green). Plasma membrane (PM) was stained withImage-iT (red) and nuclei were counterstained with DAPI. (G) Immunoblotsof 2 proton pumps subunits (ATP6V0D1 and ATP6V1A) identified by massspectrometry in the IP experiments using ALMS1 as bait (FIG. S4), αPKC,GLUT4 and β-Tubulin in cellular extracts from white adipose tissue (WAT)and kidney. 50 μg total protein loaded per lane. (H) Photograph ofDuolink positive signal detected in adipocytes using antibodies againstALMS1 and ATP6. (I) Immunofluorescence pictures showing cellularlocalizations of ATP6V0D1 and ALMS1 and merged in mature adipocytes uponINS. stimulation. (J) In silico predicted binding sites of TBC1D4 (red)and PKC (yellow) which are only 20 Angstroms away from each other in theALMS1 3D structure. (K-L) Immunodetections of αPKC, TBC1D4 and α-Actininin immunoprecipitates using ALMS1 as bait in adipocytes cultured inabsence or presence of INS.

FIG. 6. Restoring acidification in ALMS1-deprived adipocytes reinstateglucose absorption

(A-B) Time lapse pictures were performed on either control orALMS1-deprived Acridine orange stained adipocytes stimulated with INS.(C-D) Time lapse pictures were performed on either control orALMS1-deprived Acridine orange stained adipocytes stimulated with anelectroneutral K⁺/H⁺ exchange ionophore, Nigericin (NIG.). (E) Top tobottom: Scanning electron microscopy (SEM) pictures of controladipocytes stimulated either without Ins. or with INS. or with NIG.White arrows show swelled vesicles. (F) Corresponding TransmittedElectron microscopy (TEM) pictures shown in (E) showing vesicles fusionwith the plasma membrane in presence of INS. and NIG. (G) Top to bottom:SEM pictures of ALMS1-deprived adipocytes stimulated either without INSor with INS. or with NIG. (H) Corresponding TEM pictures shown in (G)showing vesicles fusion with the plasma membrane only in presence ofNIG. (I) Photographs showing the intracellular content of 2-NBDG (green)in control mature adipocytes either in absence of INS. (top panel) orafter 30 minutes INS. stimulation (middle panel) or after 30 min. NIG.Stimulation (bottom panel). (J) Photographs showing the intracellularcontent of 2-NBDG (green) in ALMS1-deprived mature adipocytes either inabsence of INS. (top panel) or after 30 minutes INS. stimulation (middlepanel) or after 30 min. NIG. stimulation (bottom panel). Scale bars: 20μm except for F and H: 500 nm.

FIG. 7. GLUT4 trafficking requires ALMSome protein complex

(A) 3D images obtained using non-permeabilized fixated mature adipocytesstimulated with NIG. following immunodetection of GLUT4 membrane bound(green). Plasma membrane (PM) was stained with Image-iT (red) and nucleiwere counterstained with DAPI. (B) Photographs showing intracellular TGcontent 24 hrs. after NIG. treatment. (C) Schematic representation ofALMS1 cellular localization and protein partner in absence of INS.stimulation in mature adipocyte. (D) Schematic representation of ALMS1dynamics and protein partners after INS. stimulation in matureadipocyte.

FIGS. 8A-8B. Glucose absorption is triggered in absence of INS throughspecific interference of αPKC binding site in the ALMSome

(A) Photographs showing absorption of 2-NBDG in presence or absence ofINS in adipocytes infected with either CTRL lentiviral particles or αPKCdomain carrying lentiviral particles. (B) Quantification ofintracellular glucose analogue 2-NB in presence or absence of INS inadipocytes infected with either CTRL lentiviral particles or αPKC domaincarrying lentiviral particles. (n=8 per group).

FIG. 9. min-αPKC-FLAG construct characterization in adipocytes

Top panel: Immunodetection of min-αPKC-FLAG using an anti-FLAG antibodyin mature adipocytes 48 hours post lentiviral infection. 2^(nd) and3^(rd) panels: 3D image of the adipocyte showing the perinuclearlocalization of min-αPKC-FLAG. Last panel: Schematic representation ofthe experimental approaches used to assess the effect of min-αPKC-FLAGon glucose absorption.

EXAMPLES

Alström syndrome (ALMS) is a rare autosomal recessive disordercharacterized by several clinical features including obesity andearly-onset diabetes. It originates due to mutations in the ALMS1 genecoding for a protein of 460 kDa.

The function of the ALMS1 gene and how it causes the Alström syndromephenotype has hitherto been unknown, with studies into its functionbeing impeded by the extremely large size of the encoded protein and itslow levels of expression.

Alström syndrome (ALMS) is a rare monogenic childhood obesity syndromefor which there is only one causative mutated gene identified to date,the ALMS1 gene. ALMS is classified as a member of the ciliopathydisorders that includes Bardet Biedl syndrome, a group of syndromicdisorders originating from mutations in the large number of differentproteins that together play a critical role in primary cilium function.Alms1 encodes the 461 kDa ALMS1 protein that was originally described tobear a purely centriolar localization, although more recent data hasalso suggested a cytoplasmic localization of ALMS1.

ALMS is clinically identified by collective multisystem phenotypethought to reflect the ubiquitous tissue expression of ALMS1, closelymimicking many of the phenotypic features of BBS. Common clinicalfeatures of ALMS include retinal degeneration, hearing loss, childhoodobesity, early-onset type 2 diabetes mellitus (T2DM) dilatedcardiomyopathy, renal and hepatic dysfunction, hypothyroidism, shortstature, hyperlipidemia, and organ fibrosis. Children with ALMS developobesity in early childhood that is associated with early onset of T2DMat around 16 years of age with a much higher overall prevalence of earlyonset T2DM in ALMS than seen with other childhood obesity syndromesresulting in a similar body mass index (BMI) including BBS. The reasonfor this predilection for T2DM in children with ALMS that is out ofproportion to their degree of obesity has remained elusive.

The inventors investigated the role of the ALMS1 protein during theadipogenic differentiation process and found that the ALMS1 proteinexpression levels increased during adipogenesis. ALMS1 suppression, inadipogenic differentiating mesenchymal stem cells, inhibited theanti-adipogenic cascades but surprisingly was not favoring adipogenesis.

In addition, the inventors showed the ALMS1 protein complex is alsorequired in mature adipocytes for efficient GLUT4 retention in itsinsulin-responsive compartment and its ability to fuse with the plasmamembrane in response to insulin stimulation. Inactivation of ALMS1decreased the amount of glucose able to be absorbed by mature adipocytesupon insulin stimulation, therefore contributing to hyperglycaemia andthe onset of diabetes.

Previous studies in the spontaneous mutant Alms^(foz/foz) andgenetrapped Alms1knockout murine ALMS models confirmed that these mice,similarly to affected human children, develop obesity in earlyadolescence due to hyperphagia, and also exhibit impaired glucosetolerance, hyperinsulinemia and islet hypertrophy, consistent withsevere insulin resistance, although the tissue origin or mechanism forthis insulin resistance has previously not been characterised.Previously published studies of in vitro studies on the murine 3T3-L1fibroblast cell line showed that inhibition of ALMS1 gene expressionresulted in mild impairment of adipogenesis but was reported to have noeffect on the insulin signaling pathway in the resulting matureadipocytes as measured by insulin-mediated AKT phosphorylation. Thisdata led directly away from the invention presented here that Alms1 doesindeed play a critical hitherto unrecognized role in the insulinsignaling pathway and in GLUT4 mediated glucose transport.

Indeed, despite the previously published contrary data, the inventorswhen carefully studying the phenotype of the Fat Aussie murine ALMSmodel (Alms1^(foz/foz)) identified that insulin resistance in this modelpreceded rather than followed the development of obesity. They furtheridentified the adipose tissue as the specific site driving the insulinresistance and subsequent development of glucose intolerance and T2DM inALMS. They confirmed that insulin signaling in Alms1^(foz/foz)adipocytes was intact all the way down to phosphorylation of TBC1D4, thelast known member of the insulin-mediated glucose uptake pathway butthen through a subsequent series of investigations identified a proteincomplex they termed the Almsome, composed of several key proteins thatassociate with ALMS1 and which together are required for the tetheringand fusion of the GLUT4 vesicles to the adipocyte plasma membrane (PM)in response to insulin signaling. The Almsome thereby represents thehitherto unidentified ultimate step in insulin-mediated glucose uptakeinto adipocytes, with insulin resistance in ALMS due to disruption ofAlmsome function leading to failure of GLUT4 membrane fusion and therebya block to adipocyte glucose transport.

Example 1 Alms1^(foz/foz) Mice Display Severe Specific Adipose TissueInsulin Resistance Even in the Absence of Obesity Animal Husbandry

Alms1^(foz/foz) mice and Alms1^(+/+) (WT) littermates were maintained ona C57BL/6J background in the animal facility on a 12 hourly light/darkcycle. Mice had free access ad libitum to water and either normal chowcontaining 5.4% fat, energy content 12 MJ/kg (Gordon's rat and mousemaintenance pellets, Gordon's specialty stockfeeds, Australia) or highfat diet (HFD) containing 23% Fat, High Simple carbohydrate, 0.19%cholesterol, energy content 20 MJ/kg (SF03-020, Specialty feeds,Australia). Primers flanking the foz mutation were used for PCRgenotyping: forward ACA ACT TTT CAT GGC TCC AGT (SEQ ID NO:12); reverseTTG GCT CAG AGA CAG TTG AAA (SEQ ID NO: 13).

Six month old obese and young (<60 days old) nonobese Alms1^(foz/foz)mice and wildtype (WT) littermates were used to investigate what primarymetabolic impairment leads Alms1^(foz/foz) mice to develop T2DM. Sixmonth old Alms1^(foz/foz) mice were obese with an average body weight of45.5 g±1.7 g compared to 26.4 g±1.3 g for WT littermates (FIG. 1A) andas previously shown had fasting hyperglycaemia and impaired glucosetolerance with elevated HOMA scores. An insulin tolerance test (ITT)showed that unlike WT (FIG. 1B) and heterozygous littermates, glycaemiain obese Alms1^(foz/foz) mice was unresponsive to insulin administration(FIG. 1B), even when doses of insulin as high as 20 U/kg wereadministered. (FIG. 1C). Obesity of Alms1^(foz/foz) mice was due tosevere adipocyte hypertrophy (FIG. 1B) rather than the adipocytehyperplasia more typically seen in obese BBS mice. To determine what theprimary defect was that was causing the glucose intolerance inAlms1^(foz/foz) mice, young lean Alms1^(foz/foz) mice were studied toremove the confounding effect of obesity on insulin responsiveness. At 2months of age, WT and Alms1^(foz/foz) males had a similar average bodyweight of −24 g (FIG. 1D). ITT in these mice showed that neverthelessthe young nonobese Alms1^(foz/foz) males already exhibited significantlyreduced insulin responsiveness (FIG. 1E), consistent with insulinresistance preceding obesity in this model. Immunodetection of TRAP,Akt, p-AKT, GLUT4, C/EBP-a and GAPDH performed on insulin sensitivetissues namely, heart, liver, skeletal muscles and white adipose tissue(WAT) of 6-month-old non-fasted Alms1^(foz/foz) and WT showed no majordifferences in protein levels except for a consistent increase in thep-AKT to total AKT ratio in WAT, consistent with a paradoxical increaserather than reduction in activation of upstream members of the insulinsignaling pathway in glucose intolerant Alms1^(foz/foz) mice (FIG. 1G).To identify which tissues alone or together might be the primary sourceof the insulin resistance observed in Alms1^(foz/foz) mice, the tissuedistribution of insulin-mediated deoxyglucose (DOG) uptake was comparedin WT and Alms1^(foz/foz) mice. This confirmed that severely impairedDOG uptake was limited to the WAT of Alms1^(foz/foz) mice with acompensatory increase in DOG uptake into the insulin-responsivegastrocnemius and soleus muscles when compared to WT mice.

These studies demonstrate that although Alms1^(foz/foz) mice becomeobese and develop progressive T2DM with age, the major initial defectcontributing to insulin resistance and hyperglycaemia is a failure inthe absence of functional ALMS1 of adipose tissue glucose uptake inresponse to insulin signaling, with this defect predating thedevelopment of obesity.

Example 2 Silencing of Alms1 in Human Adipocytes Blocks Glucose UptakeThrough Impaired GLUT4 Cellular Sorting

Materials. From Molecular Probes, Invitrogen: Acridine Orange, Image-iT®LIVE Plasma Membrane and Nuclear Staining Labeling Kit, 2-NBDG(2-(N-7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxyglucose),Hoechst 33258 and Cell Light™ Early Endosomes-RFP* BacMam 2.0*; Catalog#: A3568, 134406, N13195, H3569 and C10587. From Lonza: AdipoRed™ AssayReagent (Catalog #: PT-7009). Lentiviral particles from Santa CruzBiotechnology, INC.: ALMS1 shRNA (h) Lentiviral Particles, TBC1D4 shRNA(h) Lentiviral Particles and Control shRNA Lentiviral Particles-A;Catalog #: sc-72345-V, sc-61654-V and sc-108080 respectively. FromTocris Biosciences: Nigericin Sodium Salt (Catalog #: 4312).

Biochemical tests. Mice were tested for insulin resistance by theinsulin tolerance test (ITT) and intraperitoneal glucose tolerance test(IPGTT). For the ITT, mice were fasted 4 hours with no access to foodbut free access to water. Mice were weighed and insulin (Humulin R, EliLilly, USA) was injected ip at 0.75 U/kg body weight in 0.9% saline forinjection (Pfizer, USA). Tail blood was obtained and the plasma glucosewas determined for each mouse using a glucometer (Optium Xceed, Abbott,USA) and blood glucose test strips (Optium point of care, Abbott, USA)at 0, 15, 30 and 60 min after insulin injection. For the IPGTT, micewere fasted 18 hours and injected at 2 mg/g body weight with D-glucose(Analar, VWR, USA) in 0.9% saline for injection. Plasma glucose wasdetermined for each mouse using a glucometer with sampling via tail veinat 0, 15, 30, 60 and 120 min after glucose injection. For plasma insulinmeasurement, blood was collected on conscious animals via cheekbleeding. After collection, blood samples were kept on ice and spun at17000 g, 10 min at 4° C. Insulin levels were assayed using a commercialultrasensitive mouse insulin ELISA kit (Crystal Chem Inc., USA). Thehomeostasis model assessment of insulin resistance (HOMA-IR) index wascalculated using individual mouse fasting insulin and fasting glucoselevels. The following formula was used:

HOMA-IR=[fasting glucose (mg/dL)×fasting insulin (μU/mL)]/405.

Cell culture. Human white visceral preadipocytes (Catalog #: C-12732;PromoCell) and human mesenchymal stem cells (Catalog #: C-12974;PromoCell) derived from healthy bone marrow were purchased. Thepreadipocytes were seeded according to manufacturer's protocol andcultured in the Preadipocyte growth medium (Catalog #: C-27410;PromoCell) to confluence. One day before inducing terminal adipogenesis,the cells were infected with specific lentiviral particles consisted ofa pool of 3 shRNAs target-specific constructs purchased from Santa CruzBiotechnology and on the next day, adipogenic differentiation wasinduced by changing the medium to the Preadipocyte DifferentiationMedium (Catalog #: C-27436; PromoCell) for 2 days. After thedifferentiation phase, the medium was finally changed to the AdipocyteNutrition medium (Catalog #: C-27438; PromoCell). For the culturewithout insulin, Adipocyte Basal Medium (Catalog #: C-2431; PromoCell)without insulin was complemented with 5 g/L of deoxyglucose, 8 μg/mLd-Biotin, 400 ng/mL Dexamethasone. For the hMSCs, they were cultured inMesenchymal Stem Cell Growth Medium (Catalog #: C-28010; PromoCell) toconfluence. hMSCs were transfected with specific siRNAs as describedabove and on the next day adipogenic differentiation was induced bychanging the medium to the MSC Adipogenic Differentiation Medium(Catalog #: C28011; Promocell).

RNA extraction, cDNA synthesis, q-PCR and Taqman. Total RNA was preparedfrom the different tissues and cells using a RiboPure™ kit (Catalog #:AM1924; Ambion) followed by a DNAse treatment with the TURBO DNA-Free™(Catalog #: AM1907; Ambion). RNA integrity was assessed by gelelectrophoresis and RNA concentration by Eppendorf Biophotometer Pluswith the Hellma® Tray Cell (Catalog #: 105.810-uvs; Hellma). Reversetranscription of 1 μg total RNA to cDNA was performed using the BioRadiScript™ cDNA synthesis kit (Catalog #: 170-8891; BioRad). Real-timequantitative polymerase chain reaction amplification was performed in aBioRad CFX96 ™ Real-Time System using the iQ™ SYBR® Green Supermix(Catalog #: 170-8886; BioRAd) and primer sets optimized for testedtargets for SYBR Green-based real-time PCR for the real-time PCR. Taqmananalysis was carried out with the specific gene assay with the Taqman®Fast Advanced Master Mix (Catalog #: 4444557; Applied Biosystems). Thenormalized fold expression of the target gene was calculated using thecomparative cycle threshold (CO method by normalizing target mRNA C_(t)to those for GAPDH using the CFX Manager Software Version 1.5 and wasverified using the Lin-Reg program.

Western blots and immunofluorescence microscopy. Male Alms1^(foz/foz)and WT littermates were anaesthetized. The following insulin sensitivetissues: liver, heart, muscle and adipose tissue were harvested anddirectly placed in RIPA buffer (Tris 50 mM, NaCl 150 mM, 0.1% SDS, 1%Triton-X100) supplemented with Complete mini protease inhibitor cocktailand PhosSTOP phosphatase inhibitor cocktail (Roche, Switzerland).Samples were sonicated and centrifuged 30 min at 17 000 g, 4° C. 30 min.Protein concentration assayed with BCA assay (Thermo Fisher Scientific,USA). Cellular proteins from cells were obtained by trichloroacetic acidprecipitation and immunoblot analyses were performed using 30-50 μgtotal protein. Specific antibody binding was visualized using theSuperSignal® West Femto Maximum Sensitivity Substrate (catalog #:Lf145954, Pierce) on a BioRad Versadoc™ Imaging System or ImageQuant LAS4000 imager (GE Healthcare, UK). Nonspecific proteins stained withPonceau S were used as loading controls to normalize the signal obtainedafter specific immunodetection of the protein of interest using theBio-Rad Quantity One program. For immunofluorescence experiments, thecells were seeded on permanox 8-wells Lab-Tek II Chamber Slide (Catalog#: 177445; NUNC). Cells were treated as indicated. Then both cells andtissues cryosections were processed for protein detection after methanolfixation and permeabilized with 0.1% Triton X-100. The microscopy slideswere mounted for detection with Vectashield Mounting Medium (Catalog #:H-1200; Vector Laboratories). To view membrane-associated proteins,cells were formalin fixated for 15 min and were directly blocked,followed by immunostaining and acquisition using an upright ZeissAxiolmager Z2 microscope. Image analysis, 3D reconstitution and TimeLapse experiments and endosomes tracking experiments were performedusing either the Zeiss AxioVision program with the corresponding 3D andTracking Zeiss modules or the Zeiss Zen 2012 imaging platform.

Fluorescence measurement. The preadipocytes were cultured in a 96 wellplate and 12 wells infected with the either ALMS1 shRNA lentiviralparticles or CTRL shRNA lentiviral particles and differentiated the nextday into mature adipocytes. 3 weeks later, the intracellulartriglycerides were stained with AdipoRed staining following themanufacturer's procedure and the fluorescence was measured on a TecanInfinite 200 quad4 monochromator (Tecan, Lyon, France) at a wavelengthof 520 nm. The generated data were then analyzed using the TecanMagellan Data Analysis software using as blank unstained adipocytes.

Co-Immunoprecipitation experiments. For the co-immunoprecipitationexperiments, we used the Dynabeads® Antibody Coupling kit (Catalog #:143.11D, Invitrogen) in combination with the Dynabeads®co-immunoprecipitation kit (Catalog #: 143.21D, Invitrogen). The hMSCswere cultured to confluence and adipogenic differentiation was triggeredby medium change. 7 days after adipogenic differentiation was initiatedby medium change, the adipocytes, cultured with our without Ins. 24hours prior to lysis, were lysed under native conditions and usedaccording to the kit. After immunoprecipitation and release from thebeads, the samples were loaded on a NuPage 3-8% TrisAcetate Gel (Catalog#: EA0375BOX, Invitrogen) with a Hi Mark™ Prestained HMW ProteinStandard (Catalog #: LC5699, Invitrogen).

Protein preparation and identification by mass spectrometry. In geldigestion: The gel digestion procedure was carried out as described byRabilloud et al. (ref). Preparation of the gel pieces before trypsindigestion was performed by a liquid handler robot (QuadZ215, GilsonInternational, France). Briefly, gel bands were washed alternately with100 μl of 25 mM NH₄HCO₃ and then 100 μl of 50% acetonitrile (ACN) (3 minwash under shaking and the liquid was discarded before addition of thenext solvent). This hydrating/dehydrating cycle was repeated twice andthe pieces of gel were dried for 20 min before reduction (10 mM DTT/25mM NH₄HCO₃ buffer at 56° C. for 45 min) and alkylation (25 mMiodoacetamide/25 mM NH₄HCO₃ buffer for 45 min, room temperature).Afterwards, gel spots were again washed with 3 cycles of 25 mMNH₄HCO₃/ACN alternately. Following 20 min drying step, the gel pieceswere rehydrated by three volumes of trypsin (Promega, V5111), 12.5ng/μ1/in 25 mM NH₄HCO₃ buffer (freshly diluted) and incubated overnightat room temperature. Tryptic peptides were extracted from gel byvigorous shaking for 30 min in adapted volume of 35% H₂O/60% ACN/5%HCOOH and a 15 min sonication step.

MALDI-TOF (/TOF) mass spectrometry and database search. MALDI massmeasurement was carried out on an Autoflex III Smartbeam(Bruker-Daltonik GmbH, Bremen, Germany) matrix-assisted laserdesorption/ionization time-of-flight mass spectrometer (MALDI-TOF TOF)used in reflector positive mode. A prespotted anchorchip target (PACsystem from Bruker Daltonik, technical note TN011) with HCCA matrix wasused to analyse tryptic digests. The resulting peptide massfingerprinting data (PMF) and peptide fragment fingerprinting data (PFF)were combined by Biotools 3.2 software (Bruker Daltonik) and transferredto an intranet version of the search engine MASCOT (Matrix Science,London, UK). Variable modifications (N-term protein acetylation,methionine oxidation and cysteine carbamidomethylation) and one trypticmissed cleavage were taken into account and the peptide mass error waslimited to 50 ppm. Proteins were identified by searching data against anNCBI non-redundant protein sequence database and then submit to thehuman restricted database. In all results, the probability scores weregreater than the score fixed as significant with a p-value of 0.05.NanoLC-MSMS mass spectrometry and database search: For nanoLC-MS/MSanalysis, peptides were transferred in glass inserts, compatible withthe LC autosampler system (nanoLC-U3000, Dionex, US). The LC system wascoupled to an ESI-Q-TOF mass spectrometer (MicroTOFQ-II, Bruker,Germany). The method consisted in a 60 min run at a flow rate of 300nL/min using a gradient from two solvents: A (99.9% water: 0.1% formicacid) and B (99.92% acetonitrile: 0.08% formic acid). The systemincludes: a 300 μm×5 mm PepMap C18 used for peptides preconcentrationand a 75 μm×150 mm C18 column used for peptides elution. The TOFanalyzer was calibrated each day: data were acquired and processedautomatically using Hystar 2.8 and DataAnalysis 2.6 softwares.Consecutive searches against the NCBInr database first and then againstthe human sub-database were performed for each sample using localversions of Mascot 2.2 (MatrixScience, UK) and Proteinscape 2.0 (Bruker,Germany). False-positive rate (FPR) for protein identification wasestimated using a reverse decoy database: protein validation was doneusing a FPR below 1%. Moreover, proteins identified by only 1 peptidewere checked manually: MS/MS spectra were inspected according toconventional fragmentation rules.

In situ Proximity ligation assay (PLA). Duolink in situ PLA kit withantimouse PLUS probe and anti-rabbit MINUS probe (catalog #: 90701 and90602; OLINK Bioscience) were used in combination with the appropriateprimary antibodies according to the manufacturer's procedure. Humanprimary preadipocytes and mature adipocytes were cultured on 8-wellLab-Tek II chamber slide (Nunc) and treated as for immunofluorescencemicroscopy until the primary antibody incubation step. After washing,cells were decorated with PLA PLUS and MINUS probes (1:20 dilution) for2 hrs at 37° C. Hybridization and ligation of probes, amplification, andfinal SSC washing were performed according to the manufacturer'sprocedure. Fluorescence transfer based on protein-protein interactionwas visualized using the Duolink Detection kit 613 (OLINK Bioscience)and images were acquired.

Statistics. Statistical analyses were performed using GraphPad Prism 5software (GraphPad Software, Inc., USA). Results are shown asmeans±standard deviation. Significance of the results was determined bypaired t tests or the non-parametric Mann-Whitney U test was used forstatistical comparison of BMI and AUC data. A value of P<0.05 wasconsidered to denote statistical significance and was marked with anasterisk.

Using primary human preadipocytes as an in vitro model, the inventorslocalized ALMS1 primarily in a cytoplasmic rather than the previouslyreported centrosomal pool. ALMS1 was silenced during adipogenesis andalthough a significant decrease in the anti-adipogenic factor Pref-1 wasobserved, no major differences could be detected in expression levels ofpro-adipogenic transcription factors such as the cEBPs and PPARγ.

Following ALMS1 silencing in 2-week-old mature adipocytes, glucoseabsorption capacity was assessed using labelled glucose analogue(2-NBDG). In the absence of insulin stimulation, no 2-NBDG uptake couldbe detected in ALMS1-silenced and control mature adipocytes (FIG. 2A).On the other hand, insulin stimulation resulted in increased 2-NBDGuptake in the control human mature adipocytes (FIG. 2B, top panel) butnot ALMS1-silenced cells (FIG. 2B, bottom panel). Further to reducedglucose absorption in ALMS1-silenced adipocytes, the inventors observeda reduction in intracellular triglycerides (TG) in these cells a weeklater (FIGS. 2C-D). Of note, this reduced glucose absorption inALMS1-deficient adipocytes was not associated with decreasedphosphorylation of AKT, the downstream signaling target of insulin, aspS473-AKT levels after 30 minutes incubation with insulin were similarin both control and ALMS1-silenced human adipocytes (FIGS. 2E-F),consistent with the normal to increased levels of AKT phosphorylationpreviously observed in Alms1^(foz/foz) murine adipocytes (FIG. 1G).

The inventors next investigated the dynamics of GLUT4 in humanadipocytes in the absence of ALMS1. Insulin receptor (IR) cellularlocalization to the plasma membrane was not impaired following ALMS1silencing being detected in the vicinity of the plasma membrane (PM) inthe absence of insulin. (FIG. 2G, top panel). By contrast, inALMS1-deficient adipocytes in the absence of insulin GLUT4 lost itsperinuclear localization and was detected dispersed throughout the cellcytoplasm rather than assuming its usual perinuclear localisation. (FIG.2G, middle and bottom panels and 2H). Upon insulin stimulation, GLUT4was observed to move to the PM within the actin mesh (FIG. 21) in bothcontrol and ALMS1-silenced adipocytes. Two hours post insulinstimulation in the absence of insulin, GLUT4 was still detecteddispersed throughout the cytoplasm of the ALMS1-silenced adipocyteswhereas control adipocytes had their GLUT4 appropriately re-localized tothe perinuclear region (FIG. 2J). As there is an equilibrium betweenexocytosis and endocytosis of GLUT4 vesicles to and from the PM, theinventors checked to exclude that the impaired GLUT4 sorting inAlms1-silenced adipocytes was not due to defective GLUT4 endocytosis.Examination of dynamin, a key molecule in endocytosis, demonstrated nodifference in protein levels nor cellular localization following ALMS1silencing in adipocytes. Furthermore, the mean velocities of labelledendosomes were similar between ALMS1-silenced and control adipocytes,arguing against a defect in endocytosis being the cause of reduced GLUT4presence in the PM in response to insulin signaling.

Example 3 ALMS1 is Required for TBC1D4 Targeting to the PM in Responseto Insulin Signaling

To understand the molecular mechanism underlying the effect of ALMS1inactivation on GLUT4 localisation, the inventors identified interactingpartners of ALMS1 in human adipocytes. Immunoprecipitation (IP) usingALMS1 as the bait was performed using young mature human adipocytes (4days after differentiation trigger) followed by identification of ALMS1interacting partners by mass spectrometry. Amongst proteins wereimmunoprecipitated with ALMS1, was TBC1D4, a known AKT substrate GTPaserequired for proper retention of GLUT4 in the GLUT4 sorting vesicles(GSVs) and for the translocation of GLUT4 to the cell membrane forintracellular glucose uptake.

Example 4 Development of Structural Homology Models of ALMS1, TBC1D4 andαPKC.

As the crystal structure of Alms1 has not yet been solved, in silicostructural homology modeling was used to predict the 3D structure ofALMS1 and identify structural motifs that could bind potentialinteracting ligands (FIGS. 3A-C).

Structural Model of ALMS1. The model of ALMS1 was constructed usingfragment modeling method with the homology modelling program, Modeller.The amino acid sequence for each exon of ALMS1 was submitted toprofile-based threading algorithm available at PISRED server andsuitable templates were identified. Then those identified templateproteins were aligned with the respective exon sequences and each exonwas modeled separately using Modeller. The energy optimization andselection of models were conducted based on discrete optimized proteinenergy score. Finally, models were assembled to construct the structureof full length ALMS1 and the full-length protein was relaxed andminimized using the molecular dynamics simulation program NAMD.

Structural Model of the PTP binding domain of TBC1D4. The PTP bindingdomain of TBC1D4 is located within the first 160 residues. No reliablehomologues structure was identified to model the structure in betweenthe PTP binding domain and the Rab binding domain. Crystal structure ofthe PTP domain of murine Disabled-1(Dab-1), 1NU2 (E-value=5.2e-17),which was identified by HMM based template search at Swiss model wasused as the template for constructing the PTP binding domain of TBC1D4.

The PTP domain of TBC1D4 interacting with ALMS-1. The macromoleculardocking was performed by using the ClusPro 2.0 algorithm. Residueslocated in the interaction surface with >=0.4 angstrom overlap wereconsidered as interacting residues. Interproscan revealed that theALMS-1 contained a WD40-like domain within the first 3871 residues. WD40domain containing proteins are a family of proteins functioning asscaffolds for macro-molecular interactions.

The PTP binding domain of TBC1D4 interacting with ALMS1. Initially, thePTP binding domain and the RabGTP binding domain of TBC1D4 were dockedto the ALMS1 model using the Cluspro 2 server to determine the mostprobable site of interaction. Then both domains were docked to theirrespective interacting sites on ALMS1 using Autodock 4.2 and theirbinding affinities were calculated. Based on the affinities, the PTPbinding domain of TBC1D4 binds ALMS1 with ˜100 fold higher affinitycompared to the RabGTP binding domain. Hence, the inventors predict thatthe PTP binding domain may have a higher probability to interact withthe ALMS1 molecule compared to the RabGTP binding domain.

Modelling the PTP domain of TBC1D4. The phospho-tyrosine binding domainof TBC1D4 was modeled after identifying a suitable template from theSwiss model template identification algorithm.

Docking TBC1D4 PTP domain and RabGTP binding domain to ALMS1. Initially,the PTP binding domain and the RabGTP binding domain of TBC1D4 weredocked to ALMS1 using the Cluspro 2 server and the site of interactionwas identified. Then both domains were docked to their respectiveinteracting sites using Autodock 4.2 and their binding affinities werecalculated.

Predicted ALSM-1 residue 65, 66, 69, 72, 73, 74, 75, 76, 77, 78, 80, 87,2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, numbers, with thepotential to 2883, 2884, 2885, 2887, 2888, 2889, 2890, 2892, 2893, 2894,2895, 2897, 2909, 2910, interact with another ligand 2912, 2929, 2931,2932, 2933, 2934, 2935, 3557, 3558, 4131, 144, 145, 146, 147, 148, 149,150, 151, 193, 194, 195, 198, 199, 200, 201, 205, 208, 211, 214, 226,227, 229, 233, 234, 235, 236, 239, 242, 243, 246, 248, 249, 250, 251,252, 314, 319, 321, 986, 1341, 1344, 2269, 113, 114, 115, 116, 123, 126,127, 128, 1340, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1446, 1447,1448, 1449,1450,1451,1452,1453, 1454, 1457, 1458, 1459, 1478, 1915,1918, 1919, 1920, 1922, 1923, 1930, 2041, 2042, 2043, 2257, 2267, 2483,2484, 3866, 218, 219, 220, 221, 222, 223, 224, 277, 278, 279, 282, 285,286, 287, 288, 686, 688, 689, 690, 691, 699, 1856, 1858, 1859, 1861,1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1949,1968, 1969, 1971, 1974, 1979, 1980, 1981, 1982, 1983, 1984, 2104, 2107,2111, 2870, 2872, 2874, 2915, 3285, 3286, 3287, 793, 795, 796, 797,1285, 1314, 1408, 1409, 1422, 1423, 1425, 1426, 1427, 1430, 1431, 1671,1672, 1794, 1797, 2538, 2539, 2540, 2555, 2556, 2557, 2563, 2564, 2565,2567, 2568, 2588, 2591, 2599, 2603, 2699, 2701, 2702, 3108 Predictedresidues from ALMS1 E17, D58, S59, G62, H65, L66, Q736, T737, E738,D828, S829, T1088, D1089, mediating the interaction with A1169, Q1170,F2882, L2883, E2884 aPKC Predicted residues from aPKC F114, D116, C118,L121, N138, Q142, I145, P148, G433, E545, S562, S566, mediating theinteraction with F597, D601, W602, K604, E606, G620, T631, V664, I667ALMS1 Predicted residues from TBC1D4 G75, A76, P77, A78, R80, E81, V82,I83 mediating the interaction with ALMS1 Predicted residues from ALMS1H65, L66, S2879 mediating the interaction with TBC1D4

The homology model revealed that Alms1 assumes an apple core typestructure with a large number of bindings sites of potential ligandscentered around the core. The TBC1D4 crystal structure was similarly notsolved and hence the inventors used a homology modeling approach topredict the structure of the PTP binding domain of TBC1D4 (FIGS. 3C-D).Subsequently, in silico docking studies were performed which predictedhigh affinity binding of TBC1D4 with ALMS1 through hydrogen bonding ofTBC1D4 residues G75, A76, P77, A78, R80, E81, V82, 183 with interactingresidues H65, L66, S2879 on Alms1 (FIG. 4A). Co-localization of ALMS1and TBC1D4 was then confirmed in human adipocytes by immunofluorescencestudies (FIG. 4B). The expression levels of GLUT4, TBC1D4, and TRAP werenext tested in ALMS1-silenced adipocyte with or without insulinstimulation but no significant differences were found (FIG. 4C). Uponphosphorylation by activated AKT, phosphorylated TBC1D4 (p-TBC1D4) inadipocytes targets RAB proteins such as RAB14 and RAB10 prior to GSVsbeing targeted to the PM. However, upon insulin stimulation ofAlms1-silenced adipocytes, no difference could be detected in the levelsof TBC1D4, p-TBC1D4, RAB14 and RAB10 (FIG. 4D). The inventors nextfocused on GLUT4 cellular localization. In the absence of insulinstimulation, TBC1D4 silencing reproduced the ALMS1 silencing effect seenin mature adipocytes, i.e. a mislocalization of GLUT4 throughout thecytoplasm (FIG. 4E). In response to insulin stimulation, GLUT4 wasreleased from the perinuclear region in control adipocytes spreading-outthroughout the adipocyte cytoplasm (FIG. 4F) thereby reproducing theGLUT4 distribution pattern seen in in ALMS1 and TBC1D4-silencedadipocytes in the absence (FIG. 4E) and presence (FIG. 4F), of insulin.The inventors subsequently investigated the effect of ALMS1 silencing onthe cellular dynamics of TBC1D4 in response to insulin. In the absenceof insulin, TBC1D4 was localized to the perinuclear region in bothcontrol and ALMS1-silenced adipocytes (FIG. 4G) but notably, in responseto insulin, TBC1D4 was only transported to the PM in control but notALMS1-silenced adipocytes (FIG. 4H).

Example 5 ALMS1 Forms a Dynamic Protein Complex, the ALMSome, Requiredfor Insulin-Stimulated Glucose Transport in Human Mature Adipocytes

Although the inventors showed that ALMS1 silencing prevented TBC1D4targeting to the PM, it remained to be seen whether this impairment onits own explained the major reduction in glucose uptake observed inALMS1-deficient adipocytes. The inventors therefore compared thecellular uptake of 2-NBDG upon insulin stimulation in ALMS1 orTBC1D4-silenced or control adipocytes and found almost no 2-NBDGabsorbed in the ALMS1-silenced adipocytes compared to control adipocytes(FIGS. 5A-B), whereas whilst reduced compared to controls a substantialamount of 2-NBDG was still absorbed by TBC1D4-silenced adipocytes (FIG.5C). Subsequent GLUT4-antibody binding assays on either ALMS1 orTBC1D4-silenced or control adipocytes following 30 minutes insulinstimulation showed a high proportion of GLUT4 in the PM in control andTBC1D4-silenced adipocytes but not in ALMS1-silenced cells (FIGS. 5D-F),indicating that the secondary defect in TBC1D4 targeting to the PM inAlms1-silenced cells did not, in itself, explain the very severe defectin glucose transport and GLUT4 PM expression in ALMS1-deficient cells. Afurther examination of the Alms1 IP data revealed several subunits of Vtype ATPase proton (H⁺) pumps (A, B, D1 and G2) that the inventors thenshowed to be expressed in mature adipocytes (FIG. 5G) together withαPKC, the activating kinase of the H⁺ pumps under insulin control. Theinventors confirmed that ALMS1 was in close vicinity with the V-ATPaseH⁺ pumps in mature adipocytes in the presence of insulin both by an insitu PLA Duolink approach targeting ALMS1 and the proton pumps subunitsA1 and D1 (FIG. 5H) and also by co-immunostaining ALMS1, VATPase A1 andD1 and αPKC (FIGS. 5H, I). ALMS1 co-localized with the proton pumpsubunit V0D1 (FIG. 5I) that is integrated into the GSV membraneindicating that ALMS1 is transported in the adipocyte together with theproton pumps localized within the GSVs. Using their in silico-basedstructural model of ALMS1 interacting partners the inventors identifieda binding motif for PKC on Alms1. The binding sites for TBC1D4 and αPKCon Alms1 were in such close proximity that the model showed thatsimultaneous docking of both proteins to Alms1 was not possible due tosteric hindrance (FIG. 5J). The inventors thus hypothesized. To testtheir hypothesis that ALMS1 binding of αPKC or alternatively TBC1D4 wasunder the reciprocal control of insulin signaling in the adipocytes theinventors performed further IPs again using ALMS1 as bait but this timeusing human mature adipocytes cultured in the presence or absence ofinsulin with IPs being immunoblotted for both αPKC and TBC1D4. Theresults revealed that αPKC could only be pulled down by Alms1 anddetected by immunoblotting in extracts of adipocytes incubated in theabsence of insulin (FIG. 5K) whereas TBC1D4 was only pulled down byALMS1 and detected in extracts of adipocytes incubated in the presenceof insulin, consistent with the inventors model of reciprocalinsulin-regulated Alms1 binding (FIG. 5L).

Example 6 The ALMSome is Required for the Acidification of GSVs Prior toGLUT4 Delivery to the Plasma Membrane

While AKT-phosphorylation of TBC1D4 has been known to in some way leadto GLUT4 trafficking, the ultimate GSV-PM fusion step is an insulinregulated non-AKT dependent event that requires osmotic swelling of theGSVs under the action of the vATPase H⁺ pump. However, knowledge of theactual signal and mechanism for activation of the H⁺ pump by insulin wasmissing. The inventors tested if ALMS1 inactivation could prevent theacidification of the GSVs and therefore the chemo-osmotic-mediatedrelease of GLUT4 to the PM using the acidotrophic dye, acridine orange,which emits a green fluorescence at low concentration and an orange-redfluorescence at high concentrations in the lysosomes in which acridineorange is protonated and sequestered. In absence of insulin, noorange-red fluorescence was detected in the adipocytes. By contrast,insulin induced a rapid appearance of red color in control human matureadipocytes (FIG. 6A) but not in ALMS1-silenced adipocytes (FIG. 6B)indicating loss of insulin-mediated acidification of lysosomes inALMS1-silenced adipocytes.

The inventors next tested whether acidifying ALMS1-silenced adipocytesusing Nigericin (NIG.), an electroneutral K⁺/H⁺ exchange ionophore knownto cause osmotic swelling of the GSVs would bypass the Alms1-associateddefect in GLUT4 fusion and glucose absorption. NIG. treatment resultedin a rapid acidification of both control and ALMS1-silenced adipocytes(FIGS. 6C-D), thereby activating the swelling and fusion of theintracellular vesicles. In parallel, electron microscopy analysis showedvesicles sitting next to the PM without fusion in absence of insulin inboth control and ALMS1-silenced adipocytes (FIGS. 6E-F, top panels).Insulin treatment caused a swelling of the vesicles (FIG. 6E, middlepanels) associated with fusion of vesicles with the PM for glucoseabsorption only in the control adipocytes (FIG. 6E, middle panels) butnot in ALMS1-silenced adipocytes (FIG. 6F, middle panels). However, NIG.induced vesicular swelling and fusion with the PM in both control andALMS1-silenced adipocytes (FIGS. 6E-F, bottom panels). The NIG treatmentrestored glucose absorption in ALMS1-silenced adipocytes. While insulinhad little effect in inducing 2-NBDG absorption in ALMS1-silencedadipocytes (FIGS. 6G-H, top and middle panels), NIG not only restoredvesicle fusion but could also be shown to restore 2-NBDG absorption inthe ALMS1-silenced adipocytes to levels seen in control cells (FIGS.6G-H, bottom panels) This restored glucose transport in NIG-treatedALMS1-silenced adipocytes correlated with restored GLUT4 fusion with thePM (FIG. 7A) but not with TBC1D4 targeting to the PM (FIG. 7B); and ledto restoration of TG-filling of ALMS1-silenced adipocytes 24-hours postNIG treatment.

Example 7 Identification of Peptide Inhibitors of PKC Binding to Alms1

Once the site of binding interaction between two proteins is known, asknown in the art it is possible using knowledge of the conformation andamino acids of each protein involved in mediating the interaction, touse computational models to design peptides or small molecule drugswhich by binding in the region of the interaction site are able tosterically or otherwise hinder the binding interaction. The inventorstherefore sought to identify peptides that would inhibit the interactionof ALMS1 and αPKC or TBC1D4 using their previously described ALMS1,TBC1D4 and αPKC structural models described in Example 4. Peptidespredicted using this method to block the interaction between αPKC andALMS1 included the sequences LDSDSHYGPQHLESIDD (SEQ ID NO: 5), DSHQTEETL(SEQ ID NO: 6), QQTLPESHLP (SEQ ID NO: 7), QALLDSHLPE (SEQ ID NO: 8).PADQMTDTP (SEQ ID NO: 9), HIPEEAQKVSAV (SEQ ID NO: 10) or SCIFLEQ (SEQID NO: 11). A peptide identified using this method to block theinteraction between TBC1D4 and ALMS1 was the sequence GCGAPAAREVILVL(SEQ ID NO: 12).

Example 8 Expression of the Specific ALMS1-Interacting αPKC InteractingDomain in Mature Adipocytes Triggers Glucose Absorption in Absence ofInsulin.

Next, the inventors verified the hypothesis that insulin mediates therelease of αPKC from the ALMSome complex in order to induce glucoseabsorption. For that, they cloned the interacting domain of αPKC (SEQ IDNOs: 14 and 15) in a lentiviral vector together with a Flag-TAG. Theselected sequence was the minimum sequence of αPKC (min-αPKC-FLAG) so asto prevent sterical hindrance with the TBC1D4 interaction site on ALMS1.The expressed min-αPKC-FLAG in the adipocytes competes with theendogenous αPKC to prevent it from binding Almsome and hence favor theinsulin-mediated TBC1D4 binding to Almsome. Mature adipocytes were theninfected with either control or min-αPKC lentiviral particles to assessthe impact of min-αPKC-FLAG on glucose absorption. 48 hourspost-infection, min-αPKC-FLAG was immunodetected using an antibodyagainst the FLAG-Tag (FIG. 9). For the in vitro proof of concept, wetreated mature adipocytes as described (FIG. 9) and then incubated thetreated mature adipocytes with 2-NBDG to assess the effect ofmin-αPKC-FLAG on glucose absorption. Of interest, 2-NBDG was absorbed inmin-αPKC-FLAG treated adipocytes in absence of INS (FIG. 8A, leftcolumn) which corresponded to a 3.5 times increase compare to control(FIG. 8B). On the other hand, no significant difference was observed inpresence of INS (FIGS. 8A, right column and 8B). These data demonstratethat targeting the interaction of ALMS1 and αPKC is sufficient totrigger glucose absorption in the adipocytes irrespective of thepresence of INS.

Production of Lentiviral Vector Carrying the αPKC Domain

The ALMS1-interacting domain of human PKCα was amplified from humanHEK293 cell cDNA with N-terminal FLAG tag using Forward5′-gtacGAATTCGCCACCATGGATTACAAGGATGACGACGATAAGCTCACGGACTTCAAT TTCCTC-3′(SEQ ID NO: 16) and Reverse 5′-tagcGGATCCTCATACTGCACTCTGTAAG ATGGG-3′(SEQ ID NO: 17) primers and cloned into lentiviral vectorpCDH-EF1-MCS-IRES-puro (System Biosciences). For virus production, PKCαlentiviral vectors were transfected into 293TN cells (SystemBiosciences) along with packaging plasmids psPAX2 and pMD2.G (Addgene)with the weight ratio of 3:2:1 respectively by using Lipofectamine 2000(Life Technologies). Forty-eight hours after transfection, the culturesupernatant was harvested by centrifugation at 500×g for 10 min,followed by filtration through 0.45 μm syringe filter with PES membrane(Sartorius). The virus solution was then concentrated by adding ½ volumeof cold 30% (wt/vol) PEG6000 dissolved in 0.5M NaCl and incubatedovernight at 4° C. with occasional mixing. The mixture was thencentrifuged at 3000×g for 15 min at 4° C. Then the pellet containinglentiviral particles was resuspended in 1 mL DMEM medium and stored at−80° C. before infection of target cells.

We claim:
 1. A method of treating or delaying the progression or onsetof diabetes mellitus, insulin resistance, diabetic retinopathy, diabeticneuropathy, diabetic nephropathy, insulin resistance, hyperglycemia,obesity, and hyperinsulinaemia comprising the administration of amolecule inhibiting the binding of αPKC (Protein Kinase C alpha type) toALMS1 (Alstrom syndrome protein 1) to a subject in need of treatment. 2.The method according to claim 1, wherein said method treats or delaysthe progression or onset of type 2 diabetes mellitus in said subject. 3.The method according to claim 1, wherein the molecule does not interferewith the binding of TBC1D4 to ALMS1.
 4. The method according to claim 1,wherein the molecule is selected from the group consisting of peptidesor polypeptides or peptide mimetics, antibodies, fragments orderivatives thereof, aptamers, Spiegelmers, and chemical compounds. 5.The method according to claim 1, wherein the molecule is a peptide lessthan 50 amino acids.
 6. The method according to claim 5, wherein themolecule is a peptide comprising an amino acid sequence of a fragment ofALMS1 (SEQ ID NO: 1).
 7. The method according to claim 6, wherein themolecule is a peptide comprising an amino acid sequence of a fragment ofALMS1 including one or several of the residues which are predicted tomediate the interaction with αPKC, said residues selected from E17, D58,S59, G62, H65, L66, Q736, T737, E738, D828, 5829, T1088, D1089, A1169,Q1170, F2882, L2883, and E2884.
 8. The method according to claim 6,wherein the molecule is a peptide comprising one of the followingsequences: (SEQ ID NO: 5) LDSDSHYGPQHLESIDD; (SEQ ID NO: 6) DSHQTEETL;(SEQ ID NO: 7) QQTLPESHLP; (SEQ ID NO: 8) QALLDSHLPE; (SEQ ID NO: 9)PADQMTDTP; (SEQ ID NO: 10) HIPEEAQKVSAV; (SEQ ID NO: 11) SCIFLEQ,

and a fragment thereof comprising 6 contiguous amino acids.
 9. Themethod according to claim 5, wherein the molecule is a peptidecomprising an amino acid sequence of a fragment of αPKC (SEQ ID NO: 4).10. The method according to claim 9, wherein the molecule is a peptidecomprising an amino acid sequence of a fragment of αPKC that includesone or several of the residues which are predicted to mediate theinteraction with ALMS1 selected from F114, D116, C118, L121, N138, Q142,I145, P148, G433, E545, 5562, 5566, F597, D601, W602, K604, E606, G620,T631, V664, and
 1667. 11. An in vitro or ex vivo method for identifyingmolecules suitable for use for treating or delaying the progression oronset of diabetes mellitus, insulin resistance, diabetic retinopathy,diabetic neuropathy, diabetic nephropathy, insulin resistance,hyperglycemia, obesity, and hyperinsulinaemia, comprising contactingsaid molecules with αPKC and ALMS1 and detecting the capacity of themolecules to inhibit the binding of αPKC to ALMS1.
 12. The methodaccording to claim 11, wherein the method further comprises a step inwhich the capacity of the selected molecule to interfere with thebinding of TBC1D4 to ALMS1 is detected and the selection of themolecules which do not interfere.
 13. The method according to claim 11,wherein the binding is determined in a cellular system responsive toinsulin.
 14. The method according to claim 11, wherein the binding isdetermined in the presence and/or absence of insulin.
 15. A method oftreating or delaying the progression or onset of diabetes mellitus,insulin resistance, diabetic retinopathy, diabetic neuropathy, diabeticnephropathy, insulin resistance, hyperglycemia, obesity, andhyperinsulinaemia comprising administering a molecule that increases theexpression of ALMS1 or enhances the binding of TBC1D4 to ALMS1 to asubject in need of treatment.